For the promotion of 3R's principles of alternative methods, EU and US established ECVAM and ICCVAM for the evaluation and validation of alternative methods, respectively. This is because reliability and relevance of alternative methods should be determined by intra- and inter-laboratory validation for their regulatory acceptance. Appropriate procedures of the validation and regulatory acceptance of alternative methods were recommended by OECD in 1996. It is difficult to prepare data to satisfy the requirement by single laboratory or company. Therefore, it is recommended to establish similar institute in Japan like Japanese Center for the validation of alternative methods (JaCVAM). However, there are not enough resources in Japan. It is necessary to cooperate with Japanese Society of Alternatives to Animal Experiments (JSAAE) and Japanese industry group like Japanese Cosmetic Industry Association (JCIA). JSAAE is the only scientific community that is specified to research on alternatives. It has been contributing to the communication between scientists and animal protection groups during these 20 years. Research group supported by the Ministry of Health, Labor, and Welfare has been cooperating with JSAAE to develop and evaluate new alternative methods. We are expecting that the establishment of JaCVAM will contribute to the valid alternative test methods originated in Japan.
The origins of the Three Rs (reduction, refinement, replacement) concept of alternatives to animal experiments are briefly reviewed, and examples are given of achievements and grounds for hope in the application of the concept, as well as of causes for concern. Attention is then focused on validation, the process whereby test methods are independently evaluated for the reliability and relevance for their stated purposes. Some problems which currently confront the validation process are identified, and a recent proposal that an analogous invalidation process is needed, is discussed. Finally, emphasis is placed on the need for active collaboration in the middle ground, to advance the welfare of both humans and animals.
ECVAM was created in 1991 further to Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes which prescribes to use alternative methods whenever possible. As a service of the EU Joint Research Centre, ECVAM has pioneered the validation process and became a proactive facilitator for effective animal protection, especially in the field of regulatory toxicology. The field of alternatives is currently driven by the expectations from both cosmetics and chemicals policies: The 7th amendment to the Cosmetics Directive published in 2003 foresees to phase out animal experiments completely within 10 years. A timetable for the phasing out of the individual animal tests published by the European Commission in September 2004 had been supported by a task-force of stakeholders chaired by ECVAM. The legislation is reinforced by an immediate testing ban for finished products and for all the human health effects, for which alternative methods have been validated by ECVAM. Furthermore, a testing ban and a marketing ban, which cannot be postponed, apply in 6 years for topical and acute systemic toxicity, while the animal tests for all the other human health effects should be phased out in 10 years with a possible postponement by codecision procedure. The legislation for chemicals (REACH) is only emerging. It foresees data requirements for more than 30.000 substances produced at levels above 1 ton per year. Extensive in vivo data requirements are expected for a core of about 6.000 substances with highest production and concern. Alternative meth-ods shall first be considered throughout the testing and be predominantly used for the largest group of chemicals, namely those produced between 1-10 tons per year. A Commission proposal as to the legislation has been handed to the European Parliament and Council, and is currently under consultation. ECVAM has restructured its services directly targeting the animal tests to be replaced by in vitro and in silico approaches. Given the short time-lines to make available and implement validated methods, ECVAM is offering to steer the process of making validated alternatives available by bundling the inputs of stakeholders and by involving regulators in the process at an early stage. Steering groups com-posed of ECVAM's senior staff and complemented by external experts carry out the project management, which coordinates the various inputs. The collaboration with various stakeholders has also been reinforced. A series of activities including the initiation of three Integrated Projects have put the tailored development and validation of alternative methods on a new scale.
Developmental Neurotoxicity (DNT) is a major issue in children's health worldwide. Current meth-ods for DNT testing are complex and expensive in terms of scientific resources, time and animal use. Given the increasing number of chemicals that need to be tested and the increasing amount of information needed about them, we must look for new approaches to meet the demands for identifying developmentally neurotoxic agents with speed, reliability, and respect for animal welfare. A program developed by an international steering committee will examine in vitro and non-mammalian approaches to evaluate chemicals for their potential as developmental neurotoxicants. To date, the Steering Committee has established the program, raised initial financial support, and held a joint meeting of CAAT*-CEFIC* and ECVAM* to develop a decision approach to prioritize chemicals for immediate evaluation and began to develop an inventory of methods to measure specific end-points of interest to DNT.
The 1st open registration meeting will be held on: March 13-15, 2006 Hyatt Regency Reston Reston, Virginia (near Washington DC, Dulles Airport) (Please note the Society of Toxicology [SOT] meetings are scheduled March 5-9th, 2006 in San Diego, California) To learn more: http://caat.jhsph.edu/dnt * CAAT - Center for Alternatives to Animal Testing * CEFIC - European Chemical Industry Council * ECVAM - European Centre for the Validation of Alternative Methods
Regarding possible alternatives to skin irritation testing using animals, I will comment on the importance of comparing in vivo data with in vitro data from the viewpoint of the specific purposes and needs of product testing. I recommend an alternative method using the results of occluded human patch tests, without of the need for animal testing.
Several in vitro phototoxicity methods have been developed to assess the phototoxic potential of substances. These can be classified into two groups: the methods for screening purposes and tests focusing on the specific mechanisms of phototoxic reactions. Among these methods the 3T3 Neutral Red uptake phototoxicity test (3T3 NRU PT) was accepted as an established alternative method by ECVAM. In 2000, the EU Commission officially accepted 3T3 NRU PT for classification and labeling of chemicals to assess their acute phototoxic potential. In 2004, 3T3 NRU PT was adopted as an OECD guideline for chemicals (OECD Test Guideline TG432). This paper will describe the peer review of 3T3 NRU PT in Japan and the validation study of a new battery system proposed by Japanese industry. The paper also discusses a few recent studies using 3T3 NRU PT.
In the allergic contact hypersensitivity, the chemical penetrates into the viable layers of the epidermis and into the various cells therein. At these sites, the chemical must react with carrier protein or peptide to form a T cell epitope as well as activate Langerhans cells (LC). The reaction of haptens with proteins is either covalent or non-covalent, such as by forming a coordination complex. Meanwhile, hap-tens activate LC. Although the mechanism by which hapten activates LC is still controversial, it is accepted that LC must be activated in the initiation phase of allergic contact hypersensitivity reaction. The activated LC with augmented expression of various costimulatory molecules and increased production of proinflammatory cytokines start to migrate via the afferent lymphatics to the draining lymph nodes where they present hapten to naive T cells. To migrate to the lymph nodes, LC downregulate E-cadherin which functions in anchoring LC in the epidermis, produce MMP-9 that is required for passing through the basement membrane, and increase their expression of chemokine receptor CCR7 that induces the migration toward CCL-19 (MIP-3b) and CCL-21 (SLC). Our current and ultimate challenge is developing non-animal test methods which can predict a chemical's skin sen-sitization potential in vitro. For that purpose, it is crucial to understand the mechanism of allergic contact hypersensitivity, especially, its initiation phase. In this review, I have focused on the current understanding regarding the mechanism of activation of LC in the initiation phase.
To mimic in vivo condition for exposure to gaseous compounds, we employed the Air-Liquid Interface Culture (ALIC) of a human bronchial epithelial cell line, Calu-3, and developed a simple batch-type closed system for direct gas exposure. This system enabled relatively simple cytoxicity evaluation of various gaseous chemicals in an in vivo mimicking manner. As a preliminary evaluation of the system developed, we tested the toxicity expression of Calu-3 to benzene, tetrachloroethylene and acetone gases in terms of lactate dehydrogenase (LDH) release during 48 hours of the loading. The toxicity in ALIC exposure was higher than that in conventional exposure in the liquid phase. The reason was largely explained by numerical estimation that chemical concentrations exactly on the cell surface in the liquid culture is lower in such acute phase exposure than that in ALIC culture, in the cause of the diffusion process of molecules in the surface liquid layer. These results indicate that basic concept of the combination of ALIC of lung cells and a simple batch-type closed system is promising as a cytotoxicity test of wide ranges of gaseous compounds or samples.
During the last decade, ECVAM has supported formal validation studies on in vitro tests for predicting skin corrosivity, which included two of the in vitro tests employed human skin models, EPISKINTM and EpiDermTM. When skin models are used to evaluate skin irritancy and corrosivity, it is important that suitable chemical application procedures are utilized. We used several chemicals and evaluated their skin corrosivity using in vitro tests, evaluations using ECVAM experimental protocols and post-incubation methods that we have originally developed to predict skin irritancy, and compared the results. Vitrolife-Skin showed basic utility for corrosivity testing by both methods and it is possible to confidently predict skin corrosivity, provided that the appropriate chemical application procedures are used. In the future, human skin models will become very useful for purposes of predicting in vivo skin corrosivity of chemicals and for studying the mechanistic aspects of such corrosivity.
Osaka-Dental-University-Susceptible rats (ODUS/Odu) are a useful animal model for human periodontal disease. Through comprehensive gene expression profiling, we aimed to evaluate the utility of ODUS/Odu-derived cells as an alternative to animal models for biomedical research into human periodontal disease. Using a GeneChip Rat Expression Array containing 15923 probes, the gene expression profiles of embryonic fibroblasts obtained from 13.5-day-old embryos of ODUS/Odu or control rats were comprehensively analyzed. This profiling revealed alterations in some genes that are likely to be related to periodontal disease in ODUS/Odu, based on a comparison with genes found in databases. Osteopontin (OPN), which is involved in immune defense reactions, bone metabolism and chronic inflammation, was among the genes whose expressions were significantly altered. Realtime RT-PCR analysis showed that the OPN mRNA level was increased by more than 5.8-fold in ODUS/Odu. Moreover, the expression of CD44, a receptor for OPN, was also increased by more than 2.2-fold in ODUS/Odu. These findings support the view that OPN expression is associated with the progression of human periodontal disease. Therefore, these ODUS/Odu-derived cells may be a useful tool for studying the molecular mechanisms that underlie periodontal disease in humans.